Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is concerned with development of vaccines against HPV and other targets and elucidation of the PV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, referred to hereafter as pseudovirions. The approach involves: 1) extensive codon modification of the L1 major and L2 minor capsid proteins to remove negative regulatory elements that limit virion protein expression in replicating mammalian cells; 2) production of high copy number pseudogenomes through replication of a plasmid carrying an SV40 origin of replication in cells overexpressing SV40 Large T Antigen; and 3) efficient methods for extraction and purification of infectious particles. This methodology represents a technical breakthrough in papillomavirus research, and we have exploited this development in several ways, as outlined below. Pseudovirus production technology is being used to explore the basic features of papillomavirus virion assembly. We have determined that encapsidation of the double stranded circular genome is size-dependent, but surprisingly sequence-independent. Plasmids with no papillomavirus sequences are efficiently packaged, making these pseudovirions exceptionally flexible gene transfer vehicles. We have also determined that the capsids mature through an ordered sequence of disulfide bond formation that results in stabilization of the capsids and makes them resistant to proteolysis. In other basic virologic studies, we examined the early events in papillomavirus infection. Taking advantage of the pseudovirus approach, we determined that papillomaviruses uncoat in early endosomes and that the viral genome is subsequently directed by L2 to subnuclear domains called ND10 bodies or PODs. We further determined that co-localization of the viral genome at ND10s is required for efficient transcription of the viral genome after infection by authentic BPV1 virions. Other DNA viruses interact with ND10s early after infections, but these interactions, which do not involve capsid proteins, lead to disruption of the structures. Therefore, it has generally been found that ND10s have an antagonist function early in viral infections. Our findings are the first clear evidence that in some instances, ND10s can promote establishment of viral infection. We have developed a new papillomavirus neutralization assay based on the pseudovirus technology. This is the first high throughput papillomavirus neturalization assay and the first assay that is not severely limited by the availability of infectious capsids. It is sensitive, type-specific, and highly reproducible in a 96-well microtiter plate format. We expect this assay to greatly facilitate immune response monitoring and assessment of immune correlates of protection in the phase III VLP vaccine trials currently being conducted by the NCI, Merck, and GlaxoSmithKline. The assay should also aid in the development of second generation HPV prophylactic vaccines, and in the conduct of seroepidemiological studies. We have previously shown that display of target self-polypeptides in an ordered array on a VLP surface is effective at breaking B-cell tolerance and inducing potent antibody responses to a self-protein. In mice, display of a self-peptide on the VLPs increased IgG titers 1000-fold, compared to standard approaches involving simple linkage to T helper epitopes. High density of self-antigen display appears to be the critical determinant for abrogating B cell tolerance. We have now completed an initial study aimed at induction of auto-antibodies to the HIV co-receptor CCR5. Macaques vaccinated with VLPs displaying the N-terminus of macaque CCR5 produced high titers of auto-antibodies that bound cell surface CCR5, blocked HIV infection of cultured cells, and increased the rate of viral clearance after challenge with a CCR5-tropic SHIV. No adverse consequences of CCR5 auto-antibody production were detected in the vaccinated macaques in a two-year observation period prior to viral challenge. We have also determined that display of an amyloid beta peptide on VLPs can break B cell tolerance to amyloid beta in mice transgenic for amyloid precursor protein. A VLP-based amyloid beta vaccine might be a better candidate vaccine against Alzheimer's Disease than the vaccine recently testing in clinical trials. First, it would be expected to induce higher titer of amyloid beta antibodies, which are thought to be critical effector molecules. Second, the neural pathologies detected in a minority of recipients of the previous vaccine are thought to be result from T cell responses to the C-terminal portion of amyloid beta, while the antibodies active in amyloid beta clearance are mainly directed against the N-terminus of the protein. Therefore, in a VLP-based vaccine, the T cell help for generating the amyloid beta IgG can be directed against the linked VLPs, rather than against amyloid beta itself, thereby avoiding the adverse events associated with induction of amyloid beta T cell responses.
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